Intracellular cGMP and Ca2+ regulate vascular smooth muscle (VSM) function in health and disease. The emerging view is that intracellular Ca2+ signals are highly dynamic, and that patterning of Ca2+ signals determines VSM function. Understanding of intracellular cGMP signaling dynamics, subsequent activation of cGMP-dependent protein kinase (PKG), and its relationship to Ca2+ signals has lagged. Here, using novel cGMP biosensors and PKG inhibitors, we will provide evidence that cGMP, like Ca2+, is spatially and temporally dynamic, and dependent on multiple interrelated control mechanisms. Our approach will involve high resolution measurements of Ca2+ and cGMP, and provide an unparalleled view of the interactive control of VSM function by these messenger molecules. In Specific Aim 1 we will test the theory that membrane (pGC) and cytosolic (sGC) guanylyl cyclases engage fundamentally different patterns of cGMP formation. We propose that NO and ANP, which primarily activate sGC and pGC, respectively, create discrete pools of intracellular cGMP, and thus provide the structural basis for the functional compartmentalization of cGMP signals. We will also test the mechanisms by which PKG controls vascular tone by serving as both a negative and a positive feedback regulator for cGMP pools. These assessments have been made possible by our recent development of novel cGMP-biosensors, which allow direct measurement of cellular cGMP with high temporal and spatial resolution. In Specific Aim 2 we will elucidate the interplay between cGMP/PKG and calcium signaling. We propose that cGMP and PKG act in part through modulation of Ca2+ sparks, BKCa channels, and global calcium to regulate vascular function. In Specific Aim 3 we will determine the contributions of PKG to vascular control in vivo by studying the efficacy of PKG inhibitors to increase blood pressure and vascular resistance in the intact animal. Our approach to test the three aims of this proposal will be multidisciplinary, employing state-of-the-art techniques from physiology (high speed calcium imaging, patch clamp techniques, resistance artery myography, blood pressure and blood flow measurements), cell biology (confocal fluorescence microscopy, ratiometric fluorescence microscopy, smooth muscle cell culture), molecular biology (insect cell culture, mutagenesis, adenovirus) and biochemistry (enzyme kinetic techniques, fluorescence energy transfer, cellular protein delivery systems). This work will provide an integrated view of the factors that modulate PKG activity in vascular smooth muscle and thereby significantly enhance our understanding of arterial functions in health and disease. PUBLIC HEALTH RELEVANCE The cGMP-dependent protein kinase (PKG) is an essential regulator of cellular function in blood vessels throughout the body. This proposal seeks to ascertain the molecular mechanisms of vascular control involving PKG and its signaling partners. Understanding how blood vessels constrict and dilate is critical for the development of new strategies and therapeutic agents aimed at prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.